Only a small fraction of eukaryotic genomes encode protein or is involved in the regulation of gene expression. A far larger fraction of these genomes has little or no function and is derived from the reverse transcription of RNA. For example, at least 40% of the human genome is composed of these reverse transcripts. The gradual accumulation of such insertions over time has played a significant role in shaping the size, structure and function of our genome. The family of retrotransposable elements known as LINEs generates the protein machinery responsible for most of these insertions. One of the best model systems in which to study LINEs is R2, an element that inserts in a sequence specific manner into a fraction of the hundreds of tandemly repeated 28S rRNA genes found in all higher organisms. The high degree of sequence specificity of the R2 integration reaction has enabled detailed biochemical studies of its mechanism. A critical but poorly understood aspect of this integration mechanism is how the R2 protein binds the two ends of the R2 RNA template used for reverse transcription. One series of experiments in this proposal is a detailed study of amino acid substitutions in the R2 protein that influence its ability to bind these RNA regions. These studies will contribute to a better understanding of how LINE elements are responsible for many of the insertions that occur in a more random manner throughout the human genome. Another goal of this proposal is to understand how R2 elements are regulated. The tandemly repeated rRNA genes (rDNA locus) form the nucleolus, the site of rRNA synthesis and ribosomal subunit assembly. While each R2 insertion disrupts the function of one 28S rRNA gene, an organism can survive as long as sufficient numbers of rRNA genes remain uninserted. Thus a second series of experiments is designed to determine how organisms are able to generate high levels of rRNA from uninserted genes while minimizing the expression of R2 elements from the otherwise identical inserted rRNA genes. Transcription of inserted rRNA genes gives rise to new R2 insertion. Using methods to monitor the transcription of specific R2 elements and to position these elements within the rDNA locus, the regions of the rDNA locus that are transcribed will be defined, and these transcribed regions monitored over time to determine how they are influenced by recombination and R2 element activity. Finally, mutations in a number of genes involved in chromosome structure and rRNA gene regulation will be tested in a third series of experiments to determine whether they affect the ability of the organism to differentiate between inserted and uninserted 28S genes. These experiments should reveal new insights into how transcription of the entire rDNA locus and its R2 elements are differentially regulated. PUBLIC HEALTH RELEVANCE: Transposable elements are extremely abundant in human genomes and are responsible directly or indirectly for many mutations associated with various genetic diseases and the onset of cancer. This research studies a model system that enables detailed studies of the insertion mechanism likely to be used by the most abundant, and only active, transposable element in humans. This research also studies the regulated expression of the many ribosomal RNA genes, genes that play a key role in all aspects of cellular metabolism.